J. Microbiol. Biotechnol. (2017), 27(3), 633–643 https://doi.org/10.4014/jmb.1610.10045 Research Article Review jmb

Pro-Apoptotic Role of the Human YPEL5 Identified by Functional Complementation of a Yeast moh1Δ Ji Young Lee1, Do Youn Jun1, Ju Eun Park1, Gi Hyun Kwon1, Jong-Sik Kim2, and Young Ho Kim1*

1Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 41566, Republic of Korea 2Department of Biological Sciences, Andong National University, Andong 36729, Republic of Korea

Received: October 18, 2016 Revised: February 4, 2017 To examine the pro-apoptotic role of the human ortholog (YPEL5) of the Drosophila Yippee Accepted: February 6, 2017 , the cell viability of Saccharomyces cerevisiae mutant strain with deleted MOH1, the yeast ortholog, was compared with that of the wild-type (WT)-MOH1 strain after exposure to different apoptogenic stimulants, including UV irradiation, methyl methanesulfonate (MMS),

First published online camptothecin (CPT), heat shock, and hyperosmotic shock. The moh1Δ mutant exhibited February 7, 2017 enhanced cell viability compared with the WT-MOH1 strain when treated with lethal UV

*Corresponding author irradiation, 1.8 mM MMS, 100 μM CPT, heat shock at 50°C, or 1.2 M KCl. At the same time, the Phone: +82-53-950-5378; level of Moh1 protein was commonly up-regulated in the WT-MOH1 strain as was that of Fax: +82-53-955-5522; Ynk1 protein, which is known as a marker for DNA damage. Although the enhanced UV E-mail: [email protected] resistance of the moh1Δ mutant largely disappeared following transformation with the yeast MOH1 gene or one of the human YPEL1-YPEL5 , the transformant bearing pYES2- YPEL5 was more sensitive to lethal UV irradiation and its UV sensitivity was similar to that of the WT-MOH1 strain. Under these conditions, the UV irradiation-induced apoptotic events, such as FITC-Annexin V stainability, mitochondrial membrane potential (Δψm) loss, and metacaspase activation, occurred to a much lesser extent in the moh1Δ mutant compared with the WT-MOH1 strain and the mutant strain bearing pYES2-MOH1 or pYES2-YPEL5. These results demonstrate the functional conservation between yeast Moh1 and human YPEL5, and their involvement in mitochondria-dependent induced by DNA damage. pISSN 1017-7825, eISSN 1738-8872

Copyright© 2017 by Keywords: Apoptosis, DNA damage, Drosophila Yippee, human YPEL5, metacaspase, S. cerevisiae The Korean Society for Microbiology MOH1 and Biotechnology

Introduction YPEL family members and Drosophila Yippee, human YPEL1-YPEL4 shows 43.4-45.5% identity with Drosophila The Drosophila Yippee protein was initially identified, Yippee, and human YPEL5 shows 70.8% identity, indicating using the yeast two-hybrid method, as an intracellular YPEL5 among the human YPEL family members is the protein that interacts with a blood protein (hemoline) from most closely aligned with the Drosophila Yippee protein. the moth Hyalophora cecropia [1]. Since then, orthologs of Semiquantitative polymerase chain reaction revealed that the Drosophila Yippee protein have been found in a wide transcripts for YPEL3 and YPEL5 were ubiquitously range of , from yeast to human. During expressed in human tissues; however, those for YPEL1, comprehensive human sequence analysis, a YPEL2, and YPEL4 exhibited more restrictive expression novel gene family consisting of five members (YPEL1 patterns [2]. Immunofluorescence staining of African green through YPEL5) possessing a high with the monkey kidney fibroblast COS-7 cells showed that Ypel5 is Drosophila Yippee gene was identified [2]. Based on localized in the nucleus and centrosome at interphase, comparison of the overall sequences of human whereas it relocates to the spindle pole, mitotic spindle,

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and midbody during the M phase [2, 3]. Interestingly, constructed by deletion of the MOH1 gene, and the WT- human YPEL3, which was described as a small unstable MOH1 strain to compare their cytotoxic sensitivity to apoptotic protein in murine myeloid cells [4], has been different apoptogenic stimulants such as UV irradiation, reported to be involved in growth suppression, causing DNA-damaging drug, heat shock, and hyperosmotic shock. cellular in human cell lines, and its expression is Additionally, we have examined whether transformation regulated by the tumor suppressor protein , with lower of the moh1Δ mutant with recombinant plasmids bearing expression in several human tumors [5-7]. the MOH1 gene or one of the YPEL family genes can These previous observations raised the possibility that abrogate the elevated resistance of the mutant strain human YPEL family play an important role in the against lethal UV irradiation, due to their pro-apoptotic regulation of cell division and/or apoptosis; however, this contribution to the mitochondrial apoptotic pathway. prediction remains to be elucidated. Furthermore, it is uncertain whether the intracellular function of YPEL family Materials and Methods proteins, which was predicted in the human orthologs, can be extended to other eukaryotic species, including yeasts. Reagents, Kits, Antibodies, Cells, and Media The budding yeast Saccharomyces cerevisiae ortholog MOH1 The camptothecin (CPT) and the DNA alkylating agent methyl gene was identified as a gene (YBL049W) possessing methanesulfonate (MMS) were purchased from Sigma-Aldrich sequence similarity to the Drosophila Yippee gene when (USA). An ECL western blot kit was purchased from GE Healthcare sequencing and functional analysis were performed for a Life Sciences (UK). The FITC-Annexin V apoptosis detection kit and JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolocarbocyanine 32,560 bp segment of the left arm of S. cerevisiae chromosome iodide) were purchased from Invitrogen (USA). Anti-Ynk1 (anti- II [1, 8]. Although analysis of the phenotype of S. cerevisiae NM23-H1) was purchased from Santa Cruz Biotechnology (USA), deletion mutants can be an efficient approach for studying and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene function [9-11], there have been no reports on antibody was purchased from Thermo Scientific (USA). Rat phenotypic analysis of the mutant strain to determine antisera raised against either recombinant human YPEL5 protein MOH1 gene function. Furthermore, S. cerevisiae is a good or recombinant S. cerevisiae Moh1 protein, both of which were eukaryotic model organism for the study of many expressed as GST-fusion forms using an Escherichia coli system, fundamental biological processes, such as apoptotic cell were prepared essentially as described previously [15]. These rat death, cell cycle regulation, and DNA repair, which occur polyclonal anti-YPEL5 and anti-Moh1 antibodies were able to commonly in eukaryotic cells [12, 13]. In these contexts, we react with human YPEL5 and yeast Moh1, respectively. have decided to compare the phenotype of the moh1Δ Wild-type (WT)-MOH1 S. cerevisiae BY4741 (MATa, his3∆1, mutant strain with that of the wild-type (WT)-MOH1 leu2∆0, met15∆0, ura3∆0) and the MOH1-deletion mutant derived from S. cerevisiae BY4741 were obtained from ATCC (USA). The strain, and to determine whether the mutant phenotype WT-MOH1 strain and the moh1Δ mutant strain were cultured in can be recovered to the WT levels by complementation YPD medium containing 2% dextrose, 2% peptone, 1% yeast using plasmids expressing the human YPEL family genes. extract, and 200 μg/ml G418. The yeast strains overexpressing In a previous study, we found the human YPEL5 gene to Moh1 or YPEL1-YPEL5 were constructed by transformation of be an interesting gene, in that its mRNA expression was the yeast moh1Δ mutant with recombinant plasmid pYES2-MOH1 easily detectable in unstimulated human peripheral T cells or pYES2-YPEL1-pYES2-YPEL5. Both the WT-MOH1 strain and and then rapidly declined to undetectable level by 5 h moh1Δ mutant were transformed with empty vector plasmid following immobilized anti-CD3 activation [14]. Additionally, pYES2 and used as controls. Since the expression vector pYES2 we observed that ectopic overexpression of the YPEL5 gene contains the URA3 gene, individual transformants were selected in human cervical carcinoma HeLa cells caused a significant on synthetic complete galactose (SC-gal) solid medium (2% reduction in cell proliferation to the level of 47% of the galactose, 0.67% yeast nitrogen base w/o amino acid, 0.01% control, suggesting that YPEL5 might exert a suppressive leucine, 0.005% methionine, 0.005% , 200 μg/ml G418, and 1.8% agar). effect on cell proliferation. However, the possible role of YPEL5 in induction of apoptosis, which led to the anti- Treatment Conditions for UV Irradiation, DNA-Damaging proliferative activity, and underlying cellular and molecular Drug, Heat Shock, and Hyperosmotic Shock, and Cell Viability mechanism remain to be elucidated. Assays As an attempt to obtain direct evidence for a pro-apoptotic To compare the sensitivity of the WT-MOH1 strain and the role of human YPEL5, in the present study, we have MOH1-deletion mutant with various cytotoxic factors, the cells employed a budding yeast S. cerevisiae mutant strain, grown in YPD medium at log phase were suspended in fresh YPD

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medium (A600 = 0.4). Five microliters of 3-fold serial dilutions of AGTACATTTAC-3’) were used for the PCR. The amplified cDNA each cell suspension was plated on either YPD agar medium to fragments of the individual transcripts were cloned into the expose to UV irradiation (180 J/m2) using a UV crosslinker (CL- BamH1-EcoRI site of pYES2 (Invitrogen), resulting in pYES2- 1000; Ultra-Violet Products Ltd., USA), or YPD agar medium MOH1 and pYES2-YPEL1-pYES2-YPEL5, in which the cDNA was containing 1.8 mM MMS, 100 μg/ml CPT, or 1.2 M KCl. To placed under a strong GAL1 in sense orientation. examine their sensitivity to heat shock, the cell suspension in fresh Transformation of these recombinant plasmids into the moh1Δ

YPD medium (A600 = 0.4) was treated at 50°C for 20 min prior to mutant was performed as described previously [17]. To confirm serial 3-fold dilution and plating on YPD agar medium. the mRNA expression of individual cDNA inserts in the To estimate the sensitivity of the WT-MOH1 strain- or the transformants, RT-PCR was performed using both forward and MOH1-deletion mutant-derived transformants to lethal UV reverse primers. Additionally, for RT-PCR data normalization, irradiation, the individual cells grown in SC-gal liquid medium at the yeast housekeeping actin gene ACT1 (forward primer, log phase were resuspended in fresh SC-gal medium (A600 = 0.4). 5’-AGGTTGCTGCTTTGGTTATTGATAACGG-3’; reverse primer, Five microliters of 3-fold serial dilutions of individual cell 5’-AGCCAAGATAGAACCACCAATCCAGACG-3’) was used. suspensions was plated on SC-gal solid medium. The uncovered The PCR products were electrophoresed on a 1.2% agarose gel plates were irradiated with UV light at a diverse range of doses. containing ethidium bromide and visualized under UV light. To examine quantitatively the effect of UV irradiation on cell viability, the individual cells were appropriately diluted and Sequence Analysis and Phylogenetic Tree Construction spread on SC-gal solid medium. After the uncovered SC-gal plates The amino acid sequences of the Drosophila Yippee, S. cerevisiae were exposed to UV irradiation, the plates were incubated at 30°C Moh1, S. pombe Ypel, and related Xenopus, mouse, and human for 3 days and the colony numbers were counted to determine cell YPEL family proteins were obtained by BLAST searches of the viability. NCBI database (http://www.ncbi.nlm.nih.gov). The amino acid An equivalent cell suspension in a fresh SC-gal medium (5 ml, sequences of the Drosophila Yippee, S. cerevisiae Moh1, S. pombe

A600 = 1.2) was spread on the surface of a 100-mm-diameter Petri Ypel, Xenopus Ypel1.L-Ypel5.L, mouse Ypel1-Ypel5, and human dish and then irradiated with UV light at a diverse range of doses. YPEL1-YPEL5 were aligned using Clustal X [18], and multiple Sequentially, the cells were incubated at 30°C with shaking on a alignments were manually edited in GeneDoc (Free Software rotary shaker at 120 rpm for indicated time periods for flow Foundation, Inc., USA) to shade black for 100% conservation, gray cytometric and western blot analyses, and cell viability assays. for 80% conservation, and light gray for 60% conservation. The unrooted phylogenetic tree of the yeast Moh1 and individual Reverse Transcription-Polymerase Chain Reaction (RT-PCR) of human YPEL family members was constructed based on the Yeast MOH1 Gene and Human YPEL Family Genes, and amino acid sequence similarities using NJplot [19]. Construction of Recombinant pYES2 Plasmids PCR amplification of human YPEL1-YPEL5 cDNA from a human Yeast Cell Lysate and Western Blot Analysis T-cell cDNA library derived from Jurkat cell clone E6-1 (Stratagene, Yeast cells (5 × 107) suspended in 1× SDS sample buffer were USA) was performed using the AccuPower PCR PreMix (Bioneer, lysed by boiling for 10 min and then chilled on ice for 10 min. Korea) and specific primers. The forward primers with BamHI After centrifugation at 19,000 ×g for 20 min, the supernatant was sites and reverse primers with EcoRI sites used for PCR were collected as cell lysate. The cell lysates were subjected to YPEL1-forward, 5’-CGGATCCATGGTGAAAATGACAAAG-3’; electrophoresis on 12% SDS-polyacrylamide gels with Tris-glycine YPEL1-reverse, 5’-TGAATTCTTACTCCCAGCCATTGTC-3’; buffer, and then electrotransferred to nylon membranes. Western YPEL2-forward, 5’-CGGATCCATGGTGAAGATGACAAGA-3’; blot analysis was performed using the ECL western blotting YPEL2-reverse, 5’-AGAATTCTCAGTCCCAGCCATTGTC-3’; detection system, as described previously [20]. YPEL3-forward, 5’-CGGATCCATGGTGCGGATTTCAAA-3’; YPEL3-reverse, 5’-AGAATTCTCAGTCCCAGCCGTTGT-3’; Flow Cytometric Analysis YPEL4-forward, 5’-ATAGGATCCATGCCCAGCTGTGAC-3’; For analysis of apoptosis, yeast cells were incubated in sorbitol YPEL4-reverse, 5’-CGAATTCTCAGTCCCAGCCGTTGT-3’; buffer (0.6 M D-sorbitol, 10 mM β-mercaptoethanol, 20 mM Tris- YPEL5-forward, 5’-TCTAGGATCCATGGGCAGAATTTTC-3’; HCl, pH 7.4) containing Zymolyase 20T (20 unit/ml) at 30°C for YPEL5-reverse, 5’-ACGGAATTCTCAAGAGTTATCAGATG-3’. 15 min to prepare protoplasts. Sequentially, the protoplasts (2 × For cloning of yeast MOH1 cDNA, total RNA was purified from 106) were washed with 1× binding buffer containing 0.6 M D- the WT-MOH1 S. cerevisiae BY4741 strain as described previously sorbitol, and treated with FITC-Annexin V and propidium iodide [16]. The first-strand cDNAs were synthesized from 5 μg of total (PI) for 15 min prior to flow cytometric analysis (FACSCalibur; RNA using SuperScript II Reverse Transcriptase (Invitrogen) BD, USA). To measure intracellular metacaspase activation, yeast according to the manufacturer’s instructions. The MOH1-forward cells were stained with the CaspGLOW fluorescein active caspase primer with BamHI site (5’-AGGATCCAATATGGGATTGCGTT-3’) staining kit (Biovision Inc., USA) prior to flow cytometric analysis and MOH1-reverse primer with EcoRI site (5’-GCGGAATTCTCA according to the manufacturer’s instructions. Additionally, the cells

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stained with FITC-VAD-FMK were examined and photographed cytotoxic sensitivity of the moh1Δ mutant was compared using an LSM 700 confocal laser scanning microscope (Carl Zeiss with that of the WT-MOH1 strain under various lethal MicroImaging GmbH, Germany). The mitochondria membrane apoptogenic conditions, such as UV irradiation, DNA- potential (Δψm) loss of yeast cells was analyzed by flow damaging drug, heat shock, and hyperosmotic treatments. cytometry using JC-1 dye that has the property of aggregating When the cell viability levels of the moh1Δ mutant and the upon mitochondrial membrane polarization, forming an orange- WT-MOH1 strains following UV irradiation at lethal doses red fluorescence compound [20, 21]. Briefly, yeast cells were were compared using the 3-fold serial dilution plate assay, incubated with JC-1 dye (2 ng/ml in 1× PBS) at 30°C for 20 min, the viability of the moh1Δ mutant was much higher than and the percentage of red and green fluorescence was measured using flow cytometry. that of the WT-MOH1 strain, indicating that moh1Δ confers UV resistance (Fig. 2A). The enhanced resistance of the Statistical Analyses moh1Δ mutant compared with the WT-MOH1 strain was Unless otherwise indicated, each result in this paper is also observed following various apoptogenic treatments representative of at least three separate experiments. Statistical with 1.8 mM MMS, 100 μM CPT, heat shock at 50°C, and analysis was performed using Students t-test to evaluate the 1.2 M KCl, in which DNA damage-induced apoptosis can significance of differences between two groups, and one-way be mainly involved [22-25]. At the same time, western blot ANOVA between three and more than three groups. In all graphs, analysis revealed that the Moh1 protein level in the WT- * indicates p < 0.05 between the untreated and treated cells. All MOH1 strain was commonly enhanced at 3-6 h after data are expressed as the mean ± standard deviation (SD; for each exposure to these lethal treatments (Fig. 2B). The Ynk1 group n ≥ 3). One-way ANOVA followed by Dunnett’s multiple protein level was also elevated in common at 3-6 h. The comparison test was also used for statistical analysis using IBM yeast Ynk1 is known as a DNA damage marker, because SPSS Statistics ver. 19. Ynk1 is the yeast ortholog of human NM23-H1, possessing significant 3’-5’ exonuclease activity and nucleoside Results and Discussion diphosphatase kinase activity, which were previously required for repair of DNA damage [26, 27]. Alignment of the Amino Acid Sequences of S. cerevisiae Consequently, these previous and current results indicate Moh1 and Human YPEL Family Proteins that the Moh1 expression was up-regulated in the yeast When the amino acid sequence of S. cerevisiae Moh1 was S. cerevisiae under various lethal apoptogenic conditions to compared with those of human YPEL1-YPEL5 using the determine cytotoxic sensitivity, possibly through being Clustal X program [18], the similarity of S. cerevisiae Moh1 involved in apoptotic cell death induced by DNA damage. (GenBank P38191) showed 38.7%, 37.8%, 37.8%, 40.2%, and 31.4% identity with YPEL1 (GenBank O60688), YPEL2 Differential Sensitivities to Lethal UV Irradiation of (GenBank Q96QA6), YPEL3 (GenBank P61236), YPEL4 S. cerevisiae WT-MOH1 Strain, moh1Δ Mutant, and (GenBank Q96NS1), and YPEL5 (GenBank P62699), Mutant-Derived Transformants Expressing MOH1 Gene respectively (Fig. 1A). At the same time, human YPEL3 or Individual YPEL Family Genes shares 88.2%, 89.1%, and 79.0% identity with YPEL1, We decided to examine whether the mutant phenotype YPEL2, and YPEL4, respectively; however, YPEL5 shares can be restored to the WT phenotype by complementation 37.2%-42.0% identity with other human YPEL family using recombinant pYES2 plasmids expressing the MOH1 members. The unrooted phylogenetic relationships of gene or individual YPEL family genes. Thus, the response Drosophila Yippee protein, S. cerevisiae Moh1 protein, and of the moh1Δ mutant to a lethal dose of UV irradiation was human YPEL family proteins showed that Drosphila Yippee compared with those of the WT-MOH1 strain and the and S. cerevisiae Moh1 were grouped more closely with moh1Δ mutant-derived transformants bearing pYES2-MOH1 YPEL5 than with the other four YPEL family members or pYES2-YPEL1-pYES2-YPEL5. As evidenced by RT-PCR (Fig.1B). Consequently, these results suggest that the analysis, the moh1Δ mutant failed to express the MOH1 human ortholog of yeast Moh1 may be YPEL5, and cellular mRNA transcript, whereas the individual transformants functions of YPEL1-YPLE4 could be different from that of bearing pYES2-MOH1 or one of pYES2-YPEL1-pYES2- YPEL5; however, their functional overlapping or YPEL5 appeared to overexpress each mRNA transcript distinction from each other remains uncertain. compared with the WT-MOH1 strain (Fig.3A). Under these To examine whether the cellular function of S. cerevisiae conditions, the 3-fold serial dilution plate assay with UV Moh1 is associated with induction of apoptosis, the irradiation at lethal doses of 130, 150, and 180 J/m2 showed

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Fig. 1. Comparison of amino acid sequences (A) and unrooted phylogenetic tree (B) of Drosophila Yippee (GenBank Q9XZF0), S. cerevisiae Moh1 (GenBank P38191), S. pombe Ypel (GenBank CAB62089), Xenopus Ypel1.L (GenBank NP_001086812), Xenopus Ypel2.L (GenBank NP_001089503), Xenopus Ypel5.L (GenBank NP_001087620), and mouse and human YPEL family members. Amino acids are displayed in single-letter abbreviation after alignment for maximal identity using Clustal X [18], and their phylogenetic relationships were constructed based on the amino acid sequence similarities using NJplot [19]. Hyphens represent introduced gaps for optimum alignment. Amino acid residue numbering is shown at the end of each line. By using the GeneDoc program (Free Software Foundation, Inc., USA), conserved residues are shaded black for 100% conservation, gray for 80% conservation, and light gray for 60% conservation.

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Fig. 2. Comparison of the cell viability (A) and up-regulation of the protein levels of Moh1 and Ynk1 (B) of the WT-MOH1 strain and moh1Δ mutant strains treated with UV irradiation, methyl methanesulfonate (MMS), camptothecin (CPT), heat shock, and 1.2 M KCl. For analysis of the lethal effect of UV irradiation (180 J/m2), CPT (100 µg/ml), and heat shock (50°C for 20 min) on cell viability, the exponentially growing yeast strain was resuspended in fresh YPD medium and the 3-fold-serial dilution assay was performed as described in Materials and Methods. Equivalent cultures were prepared, harvested at the indicated time points, and subjected to western blot analysis. The yeast Ynk1 was used as the DNA damage marker.

that the UV resistance of the moh1Δ mutant was significantly counted to calculate the cell viability. Although the cell higher than that of the WT-MOH1 strain. However, the viability of the moh1Δ mutant strain appeared to be ~130- elevated UV resistance was abrogated when the mutant fold higher than that of the WT-MOH1 strain, this mutant strain was transformed with pYES2-MOH1 (Fig.3B). phenotype disappeared following transformation with Interestingly, the transformation of the mutant strain with either the yeast MOH1 gene or one of the human YPEL pYES2-YPEL1-pYES2-YPEL5 also led to abrogation of the family genes (Fig. 3C). Additionally, the transformant elevated UV resistance with no significant difference in their overexpressing the YPEL5 gene was more sensitive to abilities to restore normal UV sensitivity to the mutant lethality of UV irradiation than the other transformants strain. overexpressing one of the YPEL1-YPEL4 genes, and its For quantitative analysis of the elevated UV resistance level of UV sensitivity was similar to that of the WT-MOH1 of the moh1Δ mutant strain, the individual cells were strain. appropriately diluted, spread on SC-gal solid medium These results demonstrate that the functional role of the plates, and then exposed to lethal UV irradiation. After S. cerevisiae MOH1 gene, which is crucial for determining incubation at 30°C for 3 days, the colony numbers were UV sensitivity, can be complemented by human YPEL

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Fig. 3. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the mRNA transcripts for the yeast Moh1 and human YPEL family proteins in the WT-MOH1 strain, moh1Δ mutant strain, and mutant-derived transformants expressing the MOH1 gene or each human YPEL gene (A); UV resistance test of the yeast strains using the 3-fold serial dilution plate assay (B); and viable cell counts with the spreadplate method (C). Individual yeast strains were grown in SC-gal liquid medium, harvested at log phase, and subjected to isolation of total RNA. RT-PCR was performed as described in Materials and Methods. For RT-PCR data normalization, the yeast housekeeping actin gene ACT1 was used. To investigate the cytotoxic effect of UV irradiation, both the 3-fold serial dilution assay and the spread-plate method were used as described in Materials and Methods. After the uncovered plates were exposed to UV irradiation at the indicated doses, the plates were incubated at 30°C for 3 days before measuring cell viability. Each value is expressed as the mean ± SD (n = 3). *p < 0.05 compared with the control.

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family genes. Additionally, these results suggest that human YPEL5 gene, we examined whether yeast Moh1 and although the functional roles of human YPEL family genes human YPEL5 can play a pro-apoptotic role in the UV- overexpressed in S. cerevisiae are overlapping with the induced apoptotic pathway. yeast MOH1 gene, the YPEL5 gene is more likely to be the In accordance with the RT-PCR data, western blot human ortholog of the yeast MOH1 gene. analyses also showed that the moh1Δ mutant strain failed to express the Moh1 protein; however, the WT-MOH1 strain Pro-Apoptotic Role of Yeast Moh1 and Human YPEL5 and the transformants bearing pYES2-MOH1 and pYES2- Proteins in UV-Induced Apoptotic Pathway YPEL5 successfully expressed yeast Moh1 and human The basic apoptosis program is known to be present in YPEL5, respectively (Fig. 4A). It is noteworthy that the the yeast S. cerevisiae cells, in that the heterologous expression Moh1 level in the transformant appeared to be 24.5-fold of human Bax in yeast cells provokes several apoptotic higher than that in the WT-MOH1 strain, demonstrating events, including phosphatidylserine externalization at the overexpression of Moh1 in the transformant bearing cell surface, cytochrome c release, and DNA fragmentation, pYES2-MOH1. Under these conditions, four strains (the and that the concomitant expression of human Bcl-xL WT-MOH1 strain, moh1Δ mutant strain, and transformants prevents these effects and cell death [28-31]. Because expressing yeast MOH1 gene or human YPEL5 gene), our data indicated a contribution of the yeast MOH1 gene which were incubated for 6 h following UV irradiation as a determinant of UV sensitivity, and the functional (180 J/m2), were converted into protoplasts and analyzed complementation of yeast MOH1 gene with its orthologous by FITC-Annexin V and PI staining to detect apoptotic

Fig. 4. Western blot analyses of the S. cereviasie Moh1 protein and human YPEL5 proteins in the WT-MOH1 strain, moh1Δ mutant strain, and each transformant bearing pYES2-MOH1 or pYES2-YPEL5 (A); flow cytometric analyses of apoptotic cells using Annexin V-FITC and propidium iodide (PI) staining (B); and flow cytometric analyses of Δψm loss using JC-1 dye (C) in individual yeast strains following lethal UV exposure.

2 After exponentially growing individual yeast strains were resuspended in a fresh SC-gal medium (A600 = 0.6), exposed to UV irradiation (180 J/m ), and then incubated at 30°C with shaking for 6 h, the cells harvested were subjected to western blot and flow cytometric analyses, as described in Materials and Methods.

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cells. After treatment of the WT-MOH1 strain with UV YPEL5, the change in Δψm of the four strains following irradiation, the rate of apoptotic cells was enhanced to exposure to lethal UV irradiation was measured. The UV 34.6% (14.5% early apoptotic cells stained only with FITC- irradiation caused a significant Δψm loss in the WT-MOH1 Annexin V; 20.1% late apoptotic cells stained with both strain and transformants bearing pYES2-MOH1 or pYES2- FITC-Annexin V and PI), whereas the moh1Δ mutant strain YPEL5, with the exception of the MOH1-deletion mutant exhibited only 11.1% apoptotic cells (9.0% early apoptotic strain (Fig. 4C). Since Δψm disruption is known to be one cells; 2.1% late apoptotic cells), demonstrating that the of the initial intracellular changes leading to apoptotic cell moh1Δ mutation confers resistance to the apoptogenic effect death [33], these results suggest that the pro-apoptotic role of UV irradiation (Fig. 4B). Under these conditions, the of yeast Moh1 and human YPEL5 was involved in UV- rates of UV-induced apoptotic cells for the transformants induced Δψm disruption in yeast cells. expressing Moh1 and YPEL5 were 44.3% (21.6% early In addition, flow cytometric analysis of intracellular apoptotic cells; 22.7% late apoptotic cells) and 31.9% (16.8% metacaspase activation using FITC-VAD-FMK, which detects early apoptotic cells; 15.1% late apoptotic cells), respectively. the activation of metacaspase in yeast cells [34], revealed These results indicate that yeast Moh1 plays a pro- that the rates of cells with UV-induced metacaspase activation apoptotic role in the UV-induced apoptotic pathway, so its for the WT-MOH1 strain, moh1Δ mutant, transformant deletion mutation could confer UV resistance, and that the bearing pYES2-MOH1, and transformant bearing pYES2- pro-apoptotic role of Moh1 protein in yeast cells is YPEL5 were 67.3%, 22.1%, 85.8%, and 78.9%, respectively functionally complemented by human YPEL5 protein. (Figs.5A and 5B). Fluorescence microscopic analysis also The Δψm loss, mitochondrial cytochrome c release into showed a markedly lower rate of metacaspase activation in the cytosol, and subsequent caspase cascade activation are the moh1Δ mutant compared with WT-MOH1 strain and hallmark events of apoptosis [32]. To understand which transformants bearing pYES2-MOH1 or pYES2-YPEL5. modulator of the apoptotic pathway can be influenced by These results demonstrate that the pro-apoptotic activity of the pro-apoptotic activity of yeast Moh1 and human yeast Moh1 protein, which can be complemented by

Fig. 5. Flow cytometric and fluorescence microscopic analyses of metacaspase activation in the WT-MOH1 strain, moh1Δ mutant strain, and each transformant bearing pYES2-MOH1 or pYES2-YPEL5 following none treatment (A) and lethal UV exposure (B).

2 After exponentially growing individual yeast strains were resuspended in a fresh SC-gal medium (A600 = 0.6), exposed to UV irradiation (180 J/m ), and then incubated at 30°C with shaking for 6 h, the cells harvested were subjected to flow cytometric and fluorescence microscopic analyses, as described in Materials and Methods. The scale bar represents a length of 10 µm in each of the images.

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